This invention relates to human pancreatic elastase.
Elastase is a serine protease, capable of hydrolyzing the fibrous insoluble protein known as elastin. Elastin is a scleroprotein forming connective tissues, tendons, aortic integuments and cervical bundles of higher animals. Elastin is only slightly degraded by pepsin and trypsin.
In the course of their study on arteriosclerosis, Balo' et al observed degradation of the elastin fibers of blood vessel walls, and postulated the presence of a degrading enzyme [Balo', J and Banga, I: Schweiz Z Pathol Bacteriol, 12, 350 (1949)]. Subsequently, Banga discovered an enzyme in the pancreas which specifically degrades elastin. The enzyme was isolated in the form of crystals and named "elastase" [Banga, I: Acta Physiol Acad Sci Hung, 3, 317 (1952)].
Elastase has been confirmed to exist in the pancreas of most animals, including humans, monkeys, cats, rabbits, etc. The level is about 3.1 mg/g-pancreas in human beings, about 2.2 mg/g in bovine animals and about 10.2 mg/g in rats. A correlation is recognized between elastase activity and the age of a human being: a marked lowering in elastase activity is observed in the pancreas and plasma of males over 40 and females over 60 years [Loeven, W A and Baldwin, Maureen M: Gerontologia, 17, 170 (1971)].
In the case of patients with arteriosclerosis, the elastase activity in the pancreas was found to be markedly lower than that of healthy people, and in some cases it had completely disappeared [Balo', J, and Banga, I: Nature, 178, 310 (1956)]. Subsequent studies have also demonstrated that elastase not only degrades elastin but also promotes elastin biosynthesis.
Studies on the pharmacological action of elastase have been carried out in rats and rabbits, and have revealed the following effects:
1) inhibition of the deposition of lipids and calcium on arterial walls; PA0 2) elimination of cholesterol and calcium from arterial walls; PA0 3) selective degradation of denatured elastin; PA0 4) promotion of synthesis of elastin fibers in the arterial walls; PA0 5) lowering of serum lipids; PA0 6) improvement of lipoprotein metabolism. PA0 1) restoration of elasticity and expandability of arterial walls; PA0 2) improvement of serum lipid abnormality; PA0 3) improvement of serum lipoprotein metabolism. PA0 (a) separation of mRNA from human pancreas; PA0 (b) formation from the mRNA of a bank of cDNA in a suitable host; PA0 (c) isolation from the cDNA plasmid bank of plasmids including cDNA coding for human elastase, for example by using cDNA of human elastase I or of rat elastase II as a probe; PA0 (d) isolation of the desired cloned DNA from the plasmid. PA0 (1) inserting the DNA into an expression vector; PA0 (2) transforming a host organism; PA0 (3) culturing the transformed host under conditions resulting in expression of the DNA sequence; and PA0 (4) isolating a compound including the protein.
In clinical studies conducted on the basis of the studies mentioned above, the following effects have become apparent after oral administration of elastase:
Elastase extracted and purified from porcine pancreas was used for the above studies. However, with administration of porcine elastase to human beings, there is the risk of antibody formation due to the foreign protein, and the danger of anaphylaxis with repeated administration (Japanese Patent Application Laid-open 11180/1984). Accordingly, human elastase is preferable for human use. However it has been extremely difficult to procure human elastase in sufficient quantities from traditional sources.
At present, two kinds of human pancreatic elastase are known, elastase I and elastase II. They have not been fully characterized. For example, for pancreatic elastase II, a partial sequence of 16 amino acid residues representing 12 amino acid residues in the activation peptide and 4 amino acid residues at the N-terminal end of the elastase have been sequenced [Largman, C et al; Biochim Biophys Acta, 623, 208 (1980)]. This partial sequence is Cys Gly Asp Pro Thr Tyr Pro Pro Tyr Val Thr Arg Val Val Gly Gly.